There is an on-going need for simple, rapid and cost-effective analytical methods capable of detecting toxic substances in the environment and in biological samples. Among these toxic compounds, polychlorinated biphenyls (PCBs) have been extensively studied and proven to be harmful to humans and the environment, and consequently classified as Superfund chemicals. More recently, hydroxylated PCBs (OH-PCBs) have also been more closely examined as potentially exhibiting significant toxic health effects. Due to the toxicity and environmental and biological persistence of PCBs and OH-PCBs, it is necessary to have efficient and economical methods to detect and quantify them. Currently employed traditional analytical methods are costly, time- and labor-consuming, not amenable to field analyis, and not appropriate for extensive monitoring of target analytes in biological samples. To that end, the long-term objective of this proposal is to design and develop molecular biosensors, employing genetic engineering tools, which are selective, sensitive, portable, and inexpensive, and propose them as a viable alternative to traditional analytical methods for the detection of PCBs and OH-PCBs in biological and environmental samples. Specifically, whole-cell biosensors will be constructed that are based on bacteria engineered to harbor a plasmid containing the genes encoding for the proteins involved in the catabolic pathways of Superfund chemicals coupled to those of a signal-producing reporter protein. Additionally, the regulatory proteins of bacterial resistance operons will be redesigned to improve their binding selectivity to target analytes, and will be incorporated as the recognition elements in a new biosensing format. These redesigned proteins should be able to act like molecular switches to detect the presence of the target compounds in samples. Furthermore, the newly developed biosensing systems will be miniaturized and integrated into enabling technologies for in situ detection and field studies. As part of the enabling technologies, the transformation of bacterial wholecell sensors into spores, as long-term, highly rugged storage and transport elements, will be explored. These spore-based biosensors, as well as the molecular switches, will then be incorporated into portable miniaturized microfluidic platforms for field sensing. These biosensing systems provide with new tools for monitoring of human health and the environment, therefore, they are of high relevance for public health.